Confocal microscope apparatus

ABSTRACT

A confocal microscope apparatus comprises a first optical scanning system which obtains a scan image of a sample using a laser beam from a first laser light source, a second optical scanning system which scans specific regions of a sample with a laser beam from a second laser light source that is different from the first laser light source, thereby causing a particular phenomenon, and a beam diameter varying mechanism which can change the beam diameter of the laser beam of at least one of the first optical scanning system and the second optical scanning system. With this configuration, the apparatus further comprises an excitation light intensity distribution calculator which calculates and stores the excitation light intensity distribution along a depth direction on the sample surface from the beam diameter of the laser beam output from the beam diameter varying mechanism.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional Application of U.S. applicationSer. No. 10/393,721 filed Mar. 21, 2003, which is based upon and claimsthe benefit of priority from the prior Japanese Patent Application No.2002-89878, filed Mar. 27, 2002, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a confocal microscope apparatus which excites aspecimen which has been marked with a fluorescent dye or fluorescentprotein using the excitation wavelength, and detects fluorescenceemitted from the specimen.

2. Description of the Related Art

A scanning laser microscope has been proposed, which includes a firstoptical scanning system for obtaining a scan image of a sample and asecond optical scanning system for causing a particular phenomenon inspecific areas on the sample surface (refer to Jpn. Pat. Appln. KOKAIPublication No. 2000-275529, the entire contents of which areincorporated herein by reference). In this laser scanning microscope, aspecific area on the sample surface is irradiated using a laser lightsource and an optical path of the first optical scanning system, thusstimulating the sample or a chemical substance injected into the sample.A specific area on the sample surface which is different from theabove-mentioned area is excited using a laser light source and anoptical path of the second optical scanning system, and the fluorescenceis detected, and imaging is carried out. In the specification, unlessstated otherwise, an optical scanning system for obtaining images of asample is called a “first optical scanning system” and an opticalscanning system for causing a particular phenomenon in specific areas ofa sample is called a “second optical scanning system”.

Generally, in the confocal microscope, the focal point on the samplesurface and the conjugated focal point thereof are provided before thedetection device, and a pinhole is provided therein. Thereby, theresolution of the sample along the depth direction is 1.22 λ/NA, and asmaller confocal effect is being utilized than when a regular microscopeis used for observation. There is resolution as a result of thisconfocal effect, and thus a sharp cross sectional image (that is, animage to obtain a thin slice image along depth direction) can beobtained for the sample which is being scanned.

When the image is taken at a high speed or when a dark sample is beingused, the confocal effect is weakened by opening the pinhole (that is,enlarging a diameter of the pinhole), and the image is made bright bylowering the resolution of the fluorescence.

Thus the confocal microscope has the pinhole and decreases theresolution, and thus depth-direction information can be obtained.However, since the focal depth of the sample is determined by the fluxdiameter of the coherent light which is irradiated on the objectivelens, it is impossible to change the focal depth at the pinhole.

Meanwhile, Koehler illumination is often used as the lighting to thesample by the microscope. This Koehler illumination along the thicknessdirection of the cross section of the sample causes almost uniformexcitation.

In the conventional confocal microscope described above, when theapparatus is realized by using 2 laser scanning paths and one objectivelens, the excitation light intensity distribution along the depthdirection on the sample surface of the laser beam for sample stimulationand the laser beam for obtaining images are almost the same since onlywavelength differences is generated.

BRIEF SUMMARY OF THE INVENTION

A confocal microscope apparatus according to a first aspect of thepresent invention is characterized by comprising: a first opticalscanning system which obtains a scan image of a sample using a laserbeam from a first laser light source; a second optical scanning systemwhich scans specific regions of a sample with a laser beam from a secondlaser light source that is different from the first laser light source,thereby causing a particular phenomenon; and a beam diameter varyingmechanism which can change the beam diameter of the laser beam of atleast one of the first optical scanning system and the second opticalscanning system.

A confocal microscope apparatus according to a second aspect of thepresent invention is characterized by comprising: a first opticalscanning system which scans a sample via an objective lens withincoherent light output from an incoherent light source, and detectsfluorescence emitted from the sample via the objective lens; and asecond optical scanning system which irradiates specific regions of thesample with laser beam output from a laser light source, thereby causinga particular phenomenon, in which the first optical scanning systemfurther comprises a rotatable disk to obtain a confocal effect, thelight output from the incoherent source scans the sample via therotatable disk, and the fluorescence is detected via the rotatable disk.

A confocal microscope apparatus according to a third aspect of thepresent invention is characterized by comprising: a first optical systemwhich illuminates a sample via an objective lens with incoherent lightoutput from an incoherent light source, and detects fluorescence emittedfrom the sample via the objective lens; and a second optical scanningsystem which irradiates specific regions of a sample with a laser beamfrom a laser light source, thereby causing a particular phenomenon.

Advantages of the invention will be set forth in the description whichfollows, and in part will be obvious from the description, or may belearned by practice of the invention. Advantages of the invention may berealized and obtained by means of the instrumentalities and combinationsparticularly pointed out hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention, andtogether with the general description given above and the detaileddescription of the embodiments given below, serve to explain theprinciples of the invention.

FIG. 1 is a schematic diagram of a confocal microscope apparatusaccording to a first embodiment of the invention;

FIG. 2 is a view showing a structural example of a first beam diametervarying mechanism and a second bean diameter varying mechanism;

FIG. 3 is a schematic diagram of a confocal microscope apparatusaccording to a second embodiment of the invention;

FIG. 4 is a view showing an example of a rotatable disk used in theinvention; and

FIG. 5 is a view schematically showing a nerve tissue observation.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described with reference tothe drawings.

First Embodiment

FIG. 1 is a schematic diagram of a confocal microscope apparatusaccording to a first embodiment of the invention.

In FIG. 1, the confocal microscope apparatus comprises: a first opticalscanning system 100 for observation (or for obtaining images) whichscans a focal surface of a sample 134 with a laser beam from a firstlaser light source 101; and a second optical scanning system 200 forradiating the laser beam output from the second laser light source 201onto an optional position on the sample 134, and splitting the cagedreagent (i.e. for sample stimulation). An optical path of the firstoptical scanning system 100 and an optical path of the second opticalscanning system 200 meet at a dichroic mirror 120. As a result, thefirst optical scanning system 100 and the second optical scanning system200 share an objective lens 132.

In the first optical scanning system 100 and the second optical scanningsystem 200, the coherent light output from the first laser light source101 arrives at the dichroic mirror 120 by way of a first beam diametervarying mechanism 102 and a first optical scanning unit 104. Also, thecoherent light output from the second laser light source 201 reaches thedichroic mirror 120 by way of a beam diameter varying mechanism 202 anda second optical scanning unit 203.

In addition, the first beam diameter varying mechanism 102 and thesecond beam diameter varying mechanism 202 are connected electrically orindirectly to an excitation light intensity calculator 160. As a result,the excitation light intensity calculator 160 can obtain beam diameterinformation of the beams output from the first beam diameter varyingmechanism 102 and the second beam diameter varying mechanism 202.

The first beam diameter varying mechanism 102 and the second beamdiameter varying mechanism 202 may, as shown in FIG. 2 for example,include a plurality of mechanisms, which changes the flux diameter suchas beam expanders, on a rotatable turret. Also mechanisms, in whichoptical elements such as a plurality of lenses are combined, and theflux diameter is changed while the coherence of the laser is maintained(for example, zoom mechanism), may be adopted as the first beam diametervarying mechanism 102 and the second beam diameter varying mechanism202.

The operation of the confocal microscope apparatus according to thefirst embodiment, which has the above-described configuration, will bedescribed.

The first optical scanning system 100 and the second optical scanningsystem 200 are used for radiating a coherent light at an optional(desired) position on the sample 134. Specifically, this is as describedbelow.

That is, the flux diameter of the coherent light generated from thefirst laser light source 101 and the second laser light source 201respectively, are varied (adjusted) with the first beam diameter varyingmechanism 102 and the second beam diameter varying mechanism 202.

The light beam output from the first beam diameter varying mechanism 102passes a dichroic mirror 150, and is arbitrarily deflected to an XYdirection by each of scanning mirrors 104 a and 104 b of the firstoptical scanning unit 104. The deflected light beam is reflected at themirror 106 after passing through a relay lens 105, and is thenirradiated onto the dichroic mirror 120. Meanwhile, the light beamoutput from the second beam diameter varying mechanism 202 is suitablydeflected in an XY direction by each of scanning mirrors 203 a and 203 bof the second optical scanning unit 203. The deflected light beam passesthrough the relay lens 204 and is irradiated onto the dichroic mirror120, and the optical path is deflected at the dichroic mirror 120.

In addition, the coherent light from the dichroic mirror 120 isirradiated onto an image formation lens 130. By changing the fluxdiameters of the laser beams from the first laser light source and thesecond laser light source at the first beam diameter varying mechanism102 and the second beam diameter varying mechanism 202 with respect tothe pupil diameter of the objective lens 132, the width of theexcitation light distribution (and/or the intensity distribution) alongthe depth direction on the surface of the sample 134 corresponding toeach of the optical scanning systems can be changed.

The light beam that has passed through the image formation lens 130reaches the objective lens 132, passes through the objective lens 132and is focused on an arbitrary cross section 138 of the sample 134 whichis mounted on a stage 136. The stage 136 is movable along the XYhorizontal direction and the height direction (Z axis direction—thedirection of the arrow in FIG. 1).

As described above, when the sample 134 is being scanned, in accordancewith the application, a particular field may be scanned by each of thescanning mirrors 203 a and 203 b or it may be kept still and irradiatedin spots. Further by skipping each of the scanning mirrors 203 a and 203b momentarily, the field can be irradiated in spots at a number ofarbitrary positions from moment to moment. Meanwhile, the coherent lightgenerated from the first laser light source 101 is transmitted by thedichroic mirror 150 as described above, and it is deflected by each ofthe scanning mirrors 104 a and 104 b of the first optical scanning unit104.

When light beam is irradiated on the sample 134 by the first opticalscanning system 100, the fluorescent marker chemical is excited andfluorescence is generated.

The fluorescence from the sample 134 takes the opposite direction of theoptical path of the light irradiated on the sample 134 and passes fromthe objective lens 132 by way of the image formation lens 130, thedichroic mirror 120, the first optical scanning unit 104, the relay lens103, each of the scanning mirrors 104 a and 104 b and arrives at thedichroic mirror 150, and at the dichroic mirror 150. The fluorescence isreflected and incident to a photometry filter 140.

The light beam is incident to the photometry filter 140 and only thefluorescent wavelength from the sample 134 is selected, and the lightbeam from the sample 134 having only the fluorescence wavelength isfocused at a surface of the pinhole 144 by a lens 142. The fluorescence,which has passed through the pinhole 144, is measured by a photoelectricconversion device 146.

The excitation light intensity calculator 160 calculates the excitationlight intensity distribution on the sample surface by inputting theinformation on the beam diameter of the beam output by the first beamdiameter varying mechanism 102 and the second beam diameter varyingmechanism 202 and the performance (specification) of the objective lensbeing used at the time. It also has other functions such as outputtingvalues, which have already been stored in a memory, to interfaces suchas a computer or a display (not shown in the figure).

According to the confocal microscope apparatus of the first embodimentof the invention as mentioned above, when the sample 134 is observed andrecorded by the first optical scanning system 100, by irradiatingcoherent light on the sample 134 by the second optical scanning system200, the dynamics (chemical reactions) of sample 134 which are caused bythe coherent light irradiation by the second optical scanning system 200can be adjusted by the first optical scanning system 100.

In this case, in the first embodiment, the excitation light distributionalong the depth direction on the sample surface by the first opticalscanning system 100 and the second optical scanning system 200 areindependently set by the first beam diameter varying mechanism 102 andthe second beam diameter varying mechanism 202. Accordingly, even if theexcitation light distribution is narrow for the field of the samplebeing excited by the second optical scanning system, that is, even inthe case where a large area of the sample along the thickness directionis excited, by broadening the excitation light distribution of the firstoptical scanning system, it is possible to carry out observation.

Also, unlike the case described above, at the second optical scanningsystem, a wide field of the sample along the thickness direction isstimulated, and at the first optical scanning system, the excitationlight distribution field along the thickness direction is narrowed, andthus the cross section 138 of the sample can be observed with highresolution.

The first embodiment may be configured such that an IR pulse laser isused as the first laser light source 101, and a fluorescent image isobtained by two photon absorption. In this case, the two photonabsorption phenomenon occurs only at the position where the image isformed and theoretically the pinhole 144 is unnecessary. Also, becausethe dichroic mirror 150 can transmit the IR pulse laser, reflect thevisible fluorescence and lead it to the photoelectric converter 146,this embodiment has the property of reflecting short wavelengths. It mayalso be configured such that the beam diameter varying mechanism 102 isnot used.

As described above, by using an IR pulse laser as the first laser lightsource 101, the configuration of the first optical scanning system 100is simplified. In addition, even in the case where the first beamdiameter varying mechanism 102 is not used, the width of the excitationlight distribution along the depth direction on the sample surface ofthe optical scanning system 1 becomes narrow than the excitation lightdistribution along the depth direction of the second optical scanningsystem 200 due to the two photon absorption phenomenon. Further, in thecase where the thickness of the sample to be stimulated is to bechanged, the width of the excitation light distribution of the secondoptical scanning system 200 can be made smaller by the second beamdiameter varying mechanism 202.

Second Embodiment

A confocal microscope according to a second embodiment of the inventionis described with reference to FIG. 3. FIG. 3 is a schematic diagram ofthe confocal microscope apparatus according to the second embodiment ofthe invention. The second optical scanning system 200 of FIG. 3 is thesame as that of the first embodiment, and has been assigned the samereference numbers and thus detailed descriptions thereof are omitted.

In the second embodiment, a first optical scanning system 100′ has aincoherent light source such as a mercury light source, a halogen lightsource, or an LED light source as a light source 301. An optical lens302, a polarizing plate 303 and a polarizing beam splitter (PBS) 304 arearranged on an optical path of a light beam emitted from the lightsource 301.

A rotatable disk 305, a first image formation lens 307, a quarter waveplate 308, and objective lens 309 are arranged on a reflection opticalpath of the PBS 304, and a light beam from the light source is incidentto a sample 310, which is mounted on a stage 318, by way of these.

The rotatable disk 305 is connected to a shaft of a motor (not shown)via a rotation shaft 306, and rotates at a predetermined rotation speed.The rotatable disk 305 has linear transmit portions through which lightpasses and linear shield portions which shield light are alternatelyarranged. In addition, the line width of the shield portion is widerthan that of the transmit portion, and for example, the ratio of thewidth of the shield portion to that of the transmit portion is 1:9(refer to FIG. 4).

If the width of the portion through which light passes is W, and as isthe case with the pinhole, assuming that magnification with which thespecimen image is projected onto the disk is M, the wavelength of thelight is λ, and the numerical aperture of the objective lens is NA,W=kλM/NAwhere k is a coefficient and a value in the range of 0.5 to 1 is oftenused for k.

Also, a CCD camera 312 is arranged on the transmission optical path ofthe PBS 304 via a second image formation lens 311. A monitor 313 forobserving the image taken by the CCD camera 312 is connected to thecamera 312.

The operation of the confocal microscope of the second embodiment havingthe above configuration will be described in the following.

The light beam output from the light source 301 passes through theoptical lens 302, and at the polarizing plate 303 it is transformed tolinearly polarized light having only a predetermined polarization, andthen input into the PBS 304. The PBS 304 reflects the deflected lightbeam in the direction in which the beam has passed through thepolarizing plate and a light in a direction parallel thereto istransmitted.

The light beam reflected at the PBS 304 is input into the rotatable disk305 which rotates at a predetermined speed. The light beam passingthrough the transmit portion of the rotatable disk 305 passes throughthe first image formation lens 307 and becomes circularly polarized atthe quarter wave plate 308, and is focused on an arbitrary cross section320 of the sample 310 with the objective lens 309 to be irradiated onthe sample 310.

The light beam reflected by the sample 310 passes through the objectivelens 309, and at the quarter plate 308 it becomes linearly polarizedlight which is orthogonal to that at the time of input, thereby focusingthe image of the sample 310 on the rotatable disk 305, via the secondimaging lens 311.

The focused component of the formed image formed on the rotatable disk305 passes through the transmit portion of the rotatable disk 305. Thecomponent passing through the rotatable disk 305 is transmitted by thePBS 304, and arrives at the CCD camera 312 by way of the second imageformation lens 311. The specimen image is formed on the image formationsurface (image pickup surface).

If a particular moment when the sample 310 is being observed isconsidered, a line is projected on the sample 310 along a particulardirection as shown in FIG. 4.

In this situation, in the case where the light beam reflected from thesample 310 in this state is focused on the rotatable disk 305, a line isprojected on the rotatable disk 305 for the portion of the sample 310which is in focus. However, for the unfocused portion, the image that isprojected on the rotatable disk 305 is blurred, and thus most of theunfocused image cannot be transmitted. Accordingly, only images whichare in focus are transmitted to the rotatable disk 305.

When the rotatable disk 305 is not rotating, the situation is notchanged and the image is simply one in which the sample and the lineoverlap. However, by rotating the rotatable disk 305, the line whichincludes the transmit portion and the shield portion moves whilechanging its direction on the sample 310, and thus there is uniformity,the line image disappears and an image which is in focus can beobserved. Thus, if the rotation of the rotatable disk 305 issufficiently fast with respect to the exposure time of the CCD camera312, the focused image can be picked up by the CCD camera 312 andobserved at the monitor 313. For example, if the CCD camera 312 has a TVrate as a usual, the exposure time is 1/60 second or 1/30 second.Therefore, the number of rotations of the rotatable disk 305 during theexposure time should be about 1800 rpm at which half revolutions occur.

The excitation light distribution along the depth direction on thesurface of the sample 310 of the first optical scanning system 100′ atthis time is the same as the light distribution of Koehler illuminationof the microscope in the longitudinal direction of the slit. At thewidth direction of the slit, the distribution is the same as the secondoptical scanning system.

Accordingly, excitation light distribution along the depth direction onthe sample surface of the first optical scanning system is adistribution of which both longitudinal direction and width directiondistributions are combined. It is possible to change the excitationlight intensity distribution along the depth direction, by varying thewidth of the slit and the space between the slits of the rotatable disk305.

In the second embodiment, by detecting the dynamic change which causedreaction of the light radiated by the second optical scanning systemwhich has been shown in the first embodiment using the first opticalscanning system 100′, the excitation light distribution along the depthdirection on the surface of the first and second samples can bedifferent. Accordingly, a wider field of measurement is possible in thefirst optical scanning system 100′ than the stimulation field in thesecond optical scanning system 200.

Particularly in nervous system measurements, in order to catch movementsof the nerve which extend along the thickness direction of the sample,it is necessary to obtain the images with high speed. Usually, with theconfocal microscope apparatus, in order to increase the width of theexcitation light distribution along the depth direction on the surfaceof the sample, if the sample is extends along the thickness direction,the image cannot be captured with one measurement. As a result, as inthe second embodiment, by reducing the width of the excitation lightdistribution along the depth direction on the surface of the sample,image measurements for wider fields can be taken. Accordingly, thesecond embodiment may have a configuration in which the rotatable disk305 is omitted. Also the rotatable disk is not limited to the structureshown in FIG. 4. Provided that the confocal effect can be obtained, anyconfiguration or structure can be used. For example, the rotatable diskmay be one having pinholes formed therein, and it can be a reflectiontype rotatable disk rather than the transmit type of the above-describedembodiment.

In addition, in the second embodiment, the second beam diameter varyingmechanism 202 is not necessarily needed. However, if the secondembodiment has the second beam diameter varying mechanism 202, it ispossible to change the proportion of the first cross section and thesecond cross section, and by fine adjustment of the field for obtainingimages and the portion for stimulation, the degree of freedom of theexperiment (and/or observation) is increased. In addition, when thesecond beam diameter varying mechanism 202 is provided, it is preferablethat the excitation light intensity distribution calculator 160 isprovided as in the case of the first embodiment.

Also, in the above-described configuration, by the first opticalscanning system 100′ having an optical microscope system with Koehlerillumination, it becomes possible for the image to be obtained in awider excitation field. In this case, the rotatable disk 305 isunnecessary.

In the above-described second embodiment, the PBS 304 may be replacedwith a dichroic mirror. In this case, the light beam from the lightsource is reflected at the dichroic mirror, and the fluorescence fromthe sample passes through the dichroic mirror. Thus the optical path ofthe optical excitation system and that of the optical measurement systemcan be separated, and as a result the polarizing plate 303 isunnecessary.

Applications of the confocal microscope apparatus of each of theabove-described embodiments include for example, the application in thefield of cell research in which the cell is locally excited andreactions at the excited regions are observed.

In the method known as the uncaged method, by locally exciting the cell,the concentration of the activated material is changed. When this changein concentration is to be measured, by measuring peripheral portionsother than the locally excited regions simultaneously, the internalfunctions of the cell can be analyzed.

In the photo-bleach method, by locally exciting the cell, the excitedregions are discolored. The phenomenon is seen where due to migration ofproteins from the periphery, over time color returns to the region whichhas been discolored. Accordingly, measurements for both the locallystimulated region and the peripheral portions are necessary.

An example thereof is shown using FIG. 5. FIG. 5 is a schematic viewshowing a nerve tissue observation.

For example, when ions transmitted on an axis cylinder 3 from a cellbody 1 to a cell body 2 are observed with the caged fluorescent dyesintroduced into the cell body 1 as a probe, first a laser beam forstimulating a sample is radiated on a focal point surface 4 of the cellbody 1. Next, subsequent changes are observed with a laser beam forsample observation. However, the excitation light intensity distributionof the laser beam for sample observation 6 along the depth directionusually has the same depth as the excitation light intensitydistribution 5 of the laser beam for sample stimulation. Thus, in theprior art, the fluorescent dye which transmits the axis cylinder 3 andis not within that distribution field cannot be observed because it isnot exposed to excitation light. To the contrary, in each of theembodiments of the invention, the excitation light intensitydistribution along the depth direction, of the laser beam for samplestimulation and the laser beam for obtaining images on the surface ofthe sample are each independently varied, thus solving the problem ofthe prior art.

The inventions described in the following are extracted from theembodiments described below. The above-described embodiments do notlimit the invention. Accordingly various modifications may be madewithin the scope of the general inventive concept of the invention.

The confocal microscope apparatus according to a first aspect of thepresent invention is characterized by comprising: a first opticalscanning system which obtains a scan image of a sample using a laserbeam from a first laser light source; a second optical scanning systemwhich scans specific regions of a sample with a laser beam from a secondlaser light source that is different from the first laser light source,thereby causing a particular phenomenon; and a beam diameter varyingmechanism which can change the beam diameter of the laser beam of atleast one of the first optical scanning system and the second opticalscanning system. By combining the optical laser system and the laserscanning microscope, it becomes possible to change the width ofmeasurement by using differences in the excitation intensitydistribution along the depth direction on the surface of the sample.Specifically, this is done in the following manner.

Conventionally, when movement of the sample is being analyzed, it is ofcourse desirable for the field of excitation and the field for obtainingthe images to be different, and also for the excitation light intensitydistribution on the sample surface of the laser beam for samplestimulation along the depth direction and the excitation light intensitydistribution on the sample surface of the laser beam for obtainingimages along the depth direction to be different from each other. Inaddition, it is desirable for the width of the excitation lightintensity distribution along the depth direction to be intentionallymade small.

In the first aspect, a beam diameter varying mechanism for changing thebeam diameter of the laser beam is provided to the output exit for thelaser beam of each of the optical scanning systems. When the fluxdiameter is reduced by this beam diameter varying mechanism, thenumerical aperture of the objective lens is smaller than in the casewhere the flux diameter is large. Consequently, the width of theexcitation light intensity distribution along the depth direction on thesurface of the sample can be reduced without changing the objectivelens. Further, by providing each of the optical systems with the beamdiameter varying mechanism, the excitation light intensity distributionalong the depth direction of the sample surface of each of the opticalsystems can be changed independently. Also, the excitation lightdistribution along the depth direction on the sample surface can bechanged intentionally.

The confocal microscope apparatus according to a second aspect of thepresent invention is characterized by comprising: a first opticalscanning system which scans a sample via an objective lens withincoherent light output from an incoherent light source, and detectsfluorescence emitted from the sample via the objective lens; and asecond optical scanning system which irradiates specific regions of thesample with laser beam output from a laser light source, thereby causinga particular phenomenon, in which the first optical scanning systemfurther comprises a rotatable disk to obtain a confocal effect, thelight output from the incoherent source scans the sample via therotatable disk, and the fluorescence is detected via the rotatable disk.The optical laser system and the disk type confocal microscope apparatusare combined, so that it becomes possible to change the width formeasurement due to differences in the excitation intensity distributionalong the depth direction on the surface of the sample.

The confocal microscope apparatus according to a third aspect of thepresent invention is characterized by comprising: a first optical systemwhich illuminates a sample via an objective lens with incoherent lightoutput from an incoherent light source, and detects fluorescence emittedfrom the sample via the objective lens; and a second optical scanningsystem which irradiates specific regions of a sample with a laser beamfrom a laser light source, thereby causing a particular phenomenon. Theoptical laser system and the microscope of Koehler illumination arecombined, so that it becomes possible to change the width of measurementdue to differences in the excitation intensity distribution along thedepth direction on the surface of the sample.

Preferred embodiments of the confocal microscope described above are asdescribed in the following. Each of the embodiments may be used alone ormay used in combination.

(1) The second optical scanning system further comprises a beam diametervarying mechanism, which changes a beam diameter of the laser beam ofthe laser light source.

(2) An excitation light intensity distribution calculator whichcalculates and stores the excitation light intensity distribution alonga depth direction on the sample surface from the beam diameter of thelaser beam output from the beam diameter varying mechanism is furtherprovided.

(3) The first laser light source is an IR pulsed laser, and the beamdiameter varying mechanism is provided to the second scanning opticalsystem.

(4) In (3), a depth direction intensity distribution calculator whichcalculates an intensity distribution along a depth direction of thelaser light beam output from the beam diameter varying mechanism on thesample surface is further provided.

(5) The incoherent light source includes a lamp or an LED light source.

The observation method according to the fourth aspect of the inventionis characterized by comprising: irradiating an excitation light to asample to excite the sample; irradiating an light to cause theparticular phenomenon to a desired position; and imaging by detecting alight from the excited sample, in which said irradiating the excitationlight includes adjusting an intensity distribution of the excitationlight along a depth direction on the surface.

The observation method according to the fifth aspect of the invention ischaracterized by comprising: irradiating an excitation light to a sampleto excite the sample; irradiating an light to cause the particularphenomenon to a desired position; and imaging by detecting a light fromthe excited sample, in which said irradiating the sample includesadjusting an intensity distribution of the light to cause the particularphenomena along a depth direction on the surface.

The observation method according to the sixth aspect of the invention ischaracterized by comprising: irradiating an excitation light to a samplevia a rotatable disk to acquire a fluorescent image of the sample by adisk scanning; and irradiating an light to cause the particularphenomenon to a desired position. With this configuration, it ispreferable that the irradiating the light includes adjusting anintensity distribution of the excitation light along a depth directionon the surface.

According to the present invention, by independently changing theintensity distribution along the depth direction on the sample surfaceof the excitation light in the optical system for sample excitation andfor obtaining images, it becomes possible to do dynamic analysis ofdifferent three-dimensional spaces.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. An observation method using a confocal microscope, comprising:irradiating an excitation light to a sample, while scanning theexcitation light, to excite the sample, said excitation light having apredetermined intensity distribution along a depth direction of thesample; irradiating a stimulation light to the sample to stimulate adesired position of the sample, said stimulation light having anintensity distribution having a range along the depth direction which isnarrower than a range of the intensity distribution of the excitationlight; and detecting light emitted from the excited sample and imagingthe sample which is stimulated with the stimulation light.
 2. Theobservation method according to claim 1, wherein the imaging of thesample is carried out while changing at least one of the intensitydistribution of the excitation light and the intensity distribution ofthe stimulation light.
 3. An observation method using a confocalmicroscope, comprising: irradiating an excitation light to a sample,while scanning the excitation light, to excite the sample, saidexcitation light having a predetermined intensity distribution along adepth direction of the sample; irradiating a stimulation light to thesample to stimulate a desired position of the sample, said stimulationlight having an intensity distribution having a range along the depthdirection which is larger than a range of the intensity distribution ofthe excitation light; and detecting light emitted from the sample andimaging the sample which is stimulated with the stimulation light. 4.The observation method according to claim 3, wherein the imaging of thesample is carried out while changing at least one of the intensitydistribution of the excitation light and the intensity distribution ofthe stimulation light.
 5. A confocal microscope, comprising: anobjective lens; a first irradiation system which irradiates anexcitation light to a sample through the objective lens, while scanningthe excitation light, to excite the sample, said excitation light havinga predetermined intensity distribution along a depth direction of thesample; a second irradiation system which irradiates a stimulation lightto the sample through the objective lens to stimulate a desired positionof the sample, said stimulation light having an intensity distributionhaving a range along the depth direction which is narrower than a rangeof the intensity distribution of the excitation light; and a detectionunit which detects light emitted from the excited sample and images thesample which is stimulated with the stimulation light.
 6. The microscopeaccording to claim 5, wherein the imaging of the sample is carried outwhile changing at least one of the intensity distribution of theexcitation light and the intensity distribution of the stimulationlight.
 7. The microscope according to claim 5, further comprising: abeam diameter varying unit which varies the intensity distribution ofone of the excitation light and the stimulation light along the depthdirection by varying a beam diameter of the one of the excitation lightand the stimulation light for a pupil diameter of the objective lens. 8.The microscope according to claim 7, wherein the beam diameter varyingunit comprises a beam expander.
 9. The microscope according to claim 7,wherein the excitation light is an IR pulsed laser, and the beamdiameter varying unit is installed in the second irradiation system. 10.The microscope according to claim 5, wherein the first irradiationsystem comprises a first beam diameter varying unit which varies theintensity distribution of the excitation light along the depth directionby varying a beam diameter of the excitation light for a pupil diameterof the objective lens; and wherein the second irradiation systemcomprises a second beam diameter varying unit which varies the intensitydistribution of the stimulation light along the depth direction byvarying a beam diameter of the stimulation light for the pupil diameterof the objective lens.
 11. The microscope according to claim 5, whereinthe first irradiation system comprises a first laser light source whichirradiates laser light used as the excitation light, and the secondirradiation system comprises a second laser light source whichirradiates laser light used as the stimulation light.
 12. The microscopeaccording to claim 5, wherein the second irradiation system comprises ascanning optical unit which scans the stimulation light on the sample.13. A confocal microscope, comprising: an objective lens; a firstirradiation system which irradiates an excitation light to a samplethrough the objective lens, while scanning the excitation light, toexcite the sample, said excitation light having a predeterminedintensity distribution along a depth direction of the sample; a secondirradiation system which irradiates a stimulation light to the samplethrough the objective lens to stimulate a desired position of thesample, said stimulation light having an intensity distribution having arange along the depth direction which is larger than a range of theintensity distribution of the excitation light; and a detection unitwhich detects light emitted from the sample and images the sample whichis stimulated with the stimulation light.
 14. The microscope accordingto claim 13, wherein the imaging of the sample is carried out whilechanging at least one of the intensity distribution of the excitationlight and the intensity distribution of the stimulation light.
 15. Themicroscope according to claim 13, further comprising: a beam diametervarying unit which varies the intensity distribution of one of theexcitation light and the stimulation light along the depth direction byvarying a beam diameter of the one of the excitation light and thestimulation light for a pupil diameter of the objective lens.
 16. Themicroscope according to claim 15, wherein the beam diameter varying unitcomprises a beam expander.
 17. The microscope according to claim 15,wherein the excitation light is an IR pulsed laser, and the beamdiameter varying unit is installed in the second irradiation system. 18.The microscope according to claim 13, wherein the first irradiationsystem comprises a first beam diameter varying unit which varies theintensity distribution of the excitation light along the depth directionby varying a beam diameter of the excitation light for a pupil diameterof the objective lens; and wherein the second irradiation systemcomprises a second beam diameter varying unit which varies the intensitydistribution of the stimulation light along the depth direction byvarying a beam diameter of the stimulation light for the pupil diameterof the objective lens.
 19. The microscope according to claim 13, whereinthe first irradiation system comprises a first laser light source whichirradiates laser light used as the excitation light, and the secondirradiation system comprises a second laser light source whichirradiates laser light used as the stimulation light.
 20. The microscopeaccording to claim 13, wherein the second irradiation system comprises ascanning optical unit which scans the stimulation light on the sample.